全 文 ：植 物 分 类 学 报 45 (1): 69–74(2007) doi:10.1360/aps06020
Acta Phytotaxonomica Sinica http://www.plantsystematics.com
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Received: 13 February 2006 Accepted: 7 November 2006
* Author for correspondence. E-mail: .
Genome constitution for Musa beccarii (Musaceae) varieties
1Markku HÄKKINEN* 2Chee How TEO 3Yasmin Rofina OTHMAN
1(Botanic Garden, University of Helsinki, P. O. Box 44 (Jyrängöntie 2), FI-00014, Finland)
2(Department of Biology, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom)
3(Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia)
Abstract Musa beccarii N.W.Simmonds var. beccarii and Musa beccarii N.W.Simmonds
var. hottana Häkkinen were described earlier in Acta Phytotaxonomica et Geobotanica based
on their morphological characteristics. In order to distinguish the genomes between the two M.
beccarii varieties and M. coccinea Andrews, we have now studied them with the Inter-
Retrotransposon Amplified Polymorphism (IRAP) marker analyses. The high levels of IRAP
polymorphism detected in this study showed that M. beccarii var. beccarii and M. beccarii var.
hottana are two distinct varieties. Additional IRAP bands found in M. beccarii var. beccarii
indicated that M. beccarii var. hottana might be evolutionarily more ancient than M. beccarii
var. beccarii because LTR retrotransposons are not transpositionally removed.
Key words genome constitution, inter-retrotransposon amplified polymorphism, Musa
beccarii var. beccarii, Musa beccarii var. hottana, Musa coccinea, phylogenetic analysis.
Borneo is a large island in Southeast Asia. It is divided politically into three parts: the
kingdom of Brunei on the north central coast; the Malaysian states of Sarawak and Sabah to
the west and east; with Kalimantan of Indonesia making up the larger part to the south.
Located on the equator, it has a rainy humid equatorial climate. Borneo, being part of the
primary banana diversity center, has a large number of wild endemic banana species. As
banana plants prefer an open exposure, their growth is usually confined to rather small,
isolated populations. They consequently manifest much genetic variation. The number of
wild species of Musa L. (Linnaeus, 1753) in Borneo may now total 20, though only 17 species
have been previously described (Häkkinen, 2004, 2006). Musa beccarii N.W.Simmonds is
one of the wild species native to Borneo (Häkkinen et al., 2005). Wild Musa species are
generally grouped into four sections: Australimusa Cheesman 2n=20, Callimusa Cheesman
2n=20 including Musa beccarii 2n=18, Eumusa (Baker) Cheesman 2n=22 and Rhodochlamys
(Baker) Cheesman 2n=22 (Baker, 1893; Cheesman, 1947; Simmonds & Weatherup, 1990;
Häkkinen, 2004).
Wild populations of Musa beccarii var. beccarii have been reduced enormously due to
massive land clearing for oil palm plantations in the eastern part of Sabah. Only a few M.
beccarii populations could be found during the field study. M. beccarii var. hottana Häkkinen
is an extremely rare new variety and was only found in one location at the lower
Kinanbatangan River, Sabah.
These two varieties evolved in different directions, as adaptation to their growth
conditions. M. beccarii var. beccarii can only grow in open exposure. Under the canopy it
very soon shrivels and dies. M. beccarii var. hottana with contrary characteristics can only
grow under the canopy.
A well-known banana researcher Prof. N.W.Simmonds, who never visited Borneo,
Acta Phytotaxonomica Sinica Vol. 45 70
originally described M. beccarii from a cultivated plant, which he grew in Trinidad from
seeds imported from Sabah. He quoted that “This interesting little plant recalls Musa coccinea
Andrews in general appearance but is quite distinct from it in the deciduous basal bracts, the
large green fruits, and the long-lived male bud and, above all, in the seeds which are not of the
barrel- or top-shaped type characteristic of section Callimusa. The chromosome number,
2n=18, is new to the genus Musa. In the herbarium the plant looks like section Rhodochlamys
and I took it to be allied to M. laterita (2n=22, authors’ note) E. E. Cheesman when I first saw
specimens in the Singapore collections.” Simmonds (1956) also added “The species grows
well in Trinidad (which is rather unusual for bananas from Borneo) but sets fruit only
sporadically. The description below is based on living plants grown in Trinidad, except for
fruit characters, which are described from the Singapore specimens. So far as other characters
can be determined in the herbarium, the Trinidad and Singapore plants agree well and there is
no reasonable doubt that all the specimens cited represent but one species.” (Simmonds, 1956)
[(M. coccinea, Andrews, 1797; sect. Callimusa, Cheesman, 1947, Häkkinen, 2004; and M.
laterita, Cheesman, 1949)].
The first author has also studied the Singapore herbarium collection and agrees with
Simmonds’ observations. The first author has also written articles on the section
Rhodochlamys (Häkkinen & Sharrock, 2002) and on M. laterita (Häkkinen, 2001). M.
beccarii was treated as incertae sedis until Simmonds and Weatherup’s numerical taxonomic
analysis of wild bananas placed it in section Callimusa (Simmonds & Weatherup, 1990).
The aim of this study is to prove the distinction between M. coccinea, originating in
Guangxi, China and M. beccarii varieties, which originate in Northern Borneo.
LTR-retrotransposons are abundant in plants (Pearce et al., 1996) and propagate within
the genome via RNA intermediates by a cycle of transcription, reverse transcription, and
integration (Kumar & Bennetzen, 1999). Integration of new copies typically produces a 5–12
kb genomic insertion. These new copies are inserted and not transpositionally removed, which
facilitates phylogenetic analyses (Shimamura et al., 1997). Accumulation, fixation and
incomplete excision of retrotransposon insertions cause genomic diversification. The
ubiquitous distribution, high copy number and widespread chromosomal dispersion of
retrotransposon families provide excellent potential for developing DNA-based marker
systems (Teo et al., 2005; Ashalatha et al., 2005).
In this study, we have explored the repetitive, dispersed nature of many long terminal
repeat (LTR) retrotransposon families for characterizing genome constitutions and classifying
varieties of M. beccarii (Häkkinen et al., 2005). The retroelements’ insertional
polymorphisms were studied using banana Ty3-gypsy-like LTR (Ashalatha et al., 2005) and
barley Ty1-copia-like LTR sequences (Kalendar et al., 1999; Teo et al., 2005) as outward
primers to amplify the sequences between two LTR retrotransposons. The primers generated
specific fingerprinting patterns, which distinguish the M. coccinea, M. beccarii var. beccarii,
and M. beccarii var. hottana genomes of each species/variety and between species/varieties.
1 Material and methods
1.1 Plant materials
The first author collected fresh leaf samples of M. beccarii var. beccarii and M. beccarii
var. hottana during an expedition to Sabah in 2004. The sample of M. coccinea was from the
collection of the University of Helsinki.
Musa species and varieties studied:
Musa coccinea Andrews, accession 2001-0387 (H, Herbarium of University of Helsinki,
Finland).
No. 1 HÄKKINEN et al.: Genome constitution for Musa beccarii (Musaceae) varieties 71
Musa beccarii N.W.Simmonds var. beccarii, accession: 2004-05-18, M. Häkkinen & J.
Gisil 11 (BORH, Herbarium of Institute for Tropical Biology and Conservation, Universiti
Malaysia Sabah).
Musa beccarii N.W.Simmonds var. hottana, accession: 2004-05-20, M. Häkkinen & J.
Gisil 12 (BORH).
Leaf tissue was used for IRAP methods to generate molecular markers, which
characterize the genome constitution and diversity of species and varieties.
1.2 DNA extraction
Plant DNA was extracted using a modified CTAB method (Teo et al., 2002, 2005;
Ashalatha et al., 2005). The percentage of polyvinylpyrrolidone with molecular weight 40,000
(PVP-40, SIGMA) was increased to 6% due to the high concentration of phenolic compounds
in banana leaves.
1.3 Polymerase chain reaction
IRAP amplifications were carried out according to parameters described in Teo et al.
(2005) and Ashalatha et al. (2005). Total genomic DNA samples were diluted with sterile
H2O to 25 ng/µL. IRAP was performed in a 25 µL reaction mixture containing 50 ng DNA,
1×Promega PCR buffer, 1.5 mmol/L MgCl2, 5 pmol of each primer, 200 µmol/L dNTP mix,
1 U Taq polymerase (Promega, USA). Amplification was performed using a Tgradient
thermocycler (Whatman Biometra, Germany). The PCR reaction parameters consisted of: 95
°C, 5 min; 30 cycles of 95 °C, 30 s, annealing at the Ta specified in Table 1 for 30 s, ramp
+0.5 °C s–1 to 72 °C, and 72 °C for 2 min + 3 s extension per cycle; a final extension at 72 °C
for 10 min. PCR products were analyzed by electrophoresis on 1.5% (w/v) agarose gel and
detected by ethidium bromide staining.

Table 1 IRAP primers
Figure number Primer combination Ta (°C)
1A GyLTRev + GyLTRev 62.0
1B Sukkula LTR + Sukkula LTR 45.5
1C LTR 6150 + Nikita LTR 45.5
1D LTR 6149 + Sukkula LTR 45.5

2 Results
Inter-retrotransposon amplification polymorphism (IRAP) has been extensively used in
different plant species to study the genome diversity (Kalendar et al., 1999; Baumel et al.,
2002; Teo et al., 2005; Ashalatha et al., 2005).
Musa coccinea (2n=20), which is in the same section as both M. beccarii varieties, was
used as a reference species for Musa sectional comparison. M. laterita (2n=22), which is in a
different section (sect. Rhodochlamys), is not suitable for this type of comparison. Bartoš et
al. (2005) showed that M. laterita formed a different subgroup, from M. beccarii, with species
from sect. Eumusa and sect. Rhodochlamys in a phylogenetic analysis based on the genome
size, number of chromosomes, and number of 45S rDNA loci.
In this study, we expand the uses of banana Ty3-gypsy-like LTR (Ashalatha et al., 2005)
and barley Ty1-copia-like LTR sequences (Kalendar et al., 1999; Teo et al., 2005) on the
section Callimusa to distinguish the genome of M. beccarii var. beccarii and M. beccarii var.
hottana. The IRAP polymorphic patterns generated from Ty3-gypsy-like and Ty1-copia-like
LTRs showed clearly distinct differences between var. beccarii and var. hottana. Four LTR
primer combinations (Table 1) generated multiple fragments of defined sizes from total
Acta Phytotaxonomica Sinica Vol. 45 72
genomic DNA of var. beccarii and var. hottana and M. coccinea (Fig. 1: A–D). Species-
specific IRAP bands were observed, which enable the clear distinction of var. beccarii and
var. hottana from M. coccinea using molecular markers (arrowheads, Fig. 1: A–D). Unique
bands were observed in var. hottana, which allow var. hottana to be distinguished from var.
beccarii (arrows, Fig. 1: A–D).



Fig. 1. Polymorphism patterns of Musa beccarii var. beccarii, Musa beccarii var. hottana, and M. coccinea generated by
IRAP. A, IRAP with single GyLTRev primer. The arrowheads represent bands that enable the distinction of var. beccarii
and var. hottana from M. coccinea. B, IRAP with a single Sukkula LTR primer. The arrowheads point to bands that are
only found in M. coccinea, which distinguish M. coccinea from var. beccarii and var. hottana. C, IRAP with LTR6150 and
Nikita LTR primer combination. The arrowheads point to bands that enable the distinction of var. beccarii and var. hottana
from M. coccinea. D, IRAP with LTR6149 and Sukkula LTR primer combination. The arrowheads point to bands that
enable the distinction of var. beccarii and var. hottana from M. coccinea. The molecular weight, in base pair (bp), of each
DNA ladder is given at the left side of each figure. On the right side of each figure, the arrows point to bands that are only
found in var. hottana.

3 Discussion
The power of IRAP markers in identifying plant genomes has been demonstrated in
different plant species (Kalendar et al., 1999; Baumel et al., 2002; Teo et. al., 2005). Ty1-
copia-like and Ty3-gypsy-like retrotransposons occupy different parts of banana genomes
with different copy numbers. In addition, the non-transpositionally removal nature of LTR
retrotransposon facilitates the fingerprinting of banana genomes, which allows precise
classification of banana genomes. Using a combination of Ty1-copia-like and Ty3-gypsy-like
LTR sequences as primers in this study allows a whole genome study of Musaceae. Balint-
Kurti et al. (2000) suggested that Ty3-gypsy-like retrotransposons were introduced into Musa
genus prior to the divergence of M. acuminata Colla, M. balbisiana Colla and M. velutina
Wendl. & Drude. They were able to distinguish the A and B genomes using Ty3-gypsy-like
retrotransposons probe hybridized on HindIII-digested genomic DNA from eight cultivars of
banana with M. velutina as the control. Teo et al. (2002) showed that Ty1-copia-like
No. 1 HÄKKINEN et al.: Genome constitution for Musa beccarii (Musaceae) varieties 73
retrotransposons are present with different copy numbers in M. acuminata, M. balbisiana and
M. ornata Roxb.
The generally high levels of IRAP polymorphism with all four primer-combinations
between M. beccarii var. beccarii and M. beccarii var. hottana, together with their distinct
morphological characteristics, suggested that they are two distinct varieties (Fig. 1). This is
supported by observation of the first author during his extensive field studies in the eastern
part of Sabah, Malaysia, where var. beccarii can only grow in the open exposure whereas var.
hottana can only grow under the canopy. These polymorphisms may then be used to model
the temporal sequence of insertion events in a lineage and to establish phylogenies (Kalendar
et al., 1999).
The additional IRAP bands found in M. beccarii var. beccarii suggested that M. beccarii
var. hottana might be evolutionarily more ancient than var. beccarii. New copies of LTR
retrotransposons are not transpositionally removed (Shimamura et. al., 1997) and over time,
the accumulation, fixation and incomplete excision of retrotransposon insertions generates
more IRAP bands, which signifies genomic diversification. The association of species-
specific bands with particular genomes can be explained as the result of integration of new
retrotransposon copies after the divergence of ancestral genomes (Teo et al., 2005). The
difference in the number of chromosomes between M. coccinea (2n=20) and M. beccarii var.
beccarii (2n=18) might contribute to the presence or absence of species-specific bands in
banana genomes.
Acknowledgements The first author greatly appreciates the help and support by the staff of
Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah during the field
study in Sabah. The second author also greatly appreciates the help and support provided by
his Ph.D. supervisor, Dr. Trude SCHWARZACHER from University of Leicester, United
Kingdom. We are also grateful to Mr. Emory WALTON from California Rare Fruit Growers,
U.S.A. for proofreading this article.
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